ABSTRACT The goal of this proposal is to elucidate the mechanisms by which the novel innate immune regulator, Siah1, enhances Toll-like receptor (TLR) signaling and host defense. Siah1 is the central hub in a novel innate immunity protein interaction network that we identified using comparative genomics RNAi screens in C. elegans and mouse macrophages. This protein interaction network contains several proteins in the canonical TLR signaling pathway as well as many novel interacting proteins including Siah1. Siah1 is an E3 ubiquitin ligase that binds to the TLR signaling adaptor MyD88; we have shown that Siah1 enhances innate immunity and host defense in C. elegans and mouse macrophages. In particular, we have found that mutation of Siah1 renders C. elegans more susceptible to several bacterial pathogens. We hypothesize that Siah1 enhances the ubiquitination and subsequent proteolysis of an inhibitor of TLR signaling. We propose to determine how Siah1 regulates TLR4 signaling by (1) identifying the TLR4 signaling protein(s) whose ubiquitination and stability is regulated by Siah1, and (2) determine the mechanism used by Siah1 and its target(s) to regulate innate immunity. These studies represent a critical step towards our goal of establishing the genes in the Siah1 innate immunity protein interaction network as new therapeutic or diagnostic targets for a variety of infectious and inflammatory diseases.